It is estimated that 2% of the world's population is infected with Hepatitis C virus (HCV) and at risk of developing life-threatening liver disease. A major goal of HCV research is to understand how this virus selectively targets hepatocytes and perhaps also certain lymphocytes, but lack of appropriate experimental models has greatly limited studies of viral tropism and entry. A major advance in the field has been the recent discovery that retroviral particles pseudotyped with HCV envelope glycoproteins authentically replicate the early stages of the viral life cycle. Based on our recent studies using this novel experimental system, we postulate that HCV envelope glycoproteins bind to a receptor and subsequently to a co-receptor (CD81), which leads to internalization of viral particles and low pH-dependent fusion with intracellular membranes. Surprisingly, we discovered that HCV envelope glycoproteins cannot induce receptor-mediated, low pH-dependent fusion at the cell surface, indicating that intracellular factors critically modulate post-attachment steps of viral entry. Ongoing studies in our laboratory aim to elucidate the viral and cellular factors that regulate the unique and complex cascade of events resulting in HCV fusion with target cell membranes. Here, we propose to determine the basic mechanism of HCV internalization and trafficking to fusion-competent intracellular compartments. In the long term, we plan to identify the intracellular factors that allow HCV entry to occur in specific organelles. The entry stage of viral replication constitutes a target for neutralizing antibodies as well as pharmacologic agents. A detailed understanding of the downstream events leading to viral entry is therefore critical and may yield additional targets for preventive or therapeutic intervention. These studies will significantly advance our understanding of the mechanism by which HCV enters liver cells and will serve as a foundation for developing specific inhibitors that target this key step of viral replication.